The Actin-Based Nanomachine at the Leading Edge of Migrating Cells

Abraham, Vivek C.; Krishnamurthi, V.; Taylor, D. Lansing; Lanni, Frederick

doi:10.1016/s0006-3495(99)77018-9

Cited by 247 publications

(221 citation statements)

References 57 publications

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“…Other experiments however (22,23) suggested that the propelling force is much larger, of the order of few nN. Likewise, the force exerted by the lamellipodium on a moving cell is of the order of a few nN (24). The discrepancies described above call for direct measurements of the force generated by actin polymerization, which are carried out here.…”

mentioning

confidence: 73%

Forces generated during actin-based propulsion: A direct measurement by micromanipulation

Marcy

Prost

Carlier

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

235

208

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Dynamic actin networks generate forces for numerous types of movements such as lamellipodia protrusion or the motion of endocytic vesicles. The actin-based propulsive movement of Listeria monocytogenes or of functionalized microspheres have been extensively used as model systems to identify the biochemical components that are necessary for actin-based motility. However, quantitative force measurements are required to elucidate the mechanism of force generation, which is still under debate. To directly probe the forces generated in the process of actin-based propulsion, we developed a micromanipulation experiment. A comet growing from a coated polystyrene bead is held by a micropipette while the bead is attached to a force probe, by using a specially designed ''flexible handle.'' This system allows us to apply both pulling and pushing external forces up to a few nanonewtons. By pulling the actin tail away from the bead at high speed, we estimate the elastic modulus of the gel and measure the force necessary to detach the tail from the bead. By applying a constant force in the range of ؊1.7 to 4.3 nN, the force-velocity relation is established. We find that the relation is linear for pulling forces and decays more weakly for pushing forces. This behavior is explained by using a dimensional elastic analysis.T he growth of a polymer against a barrier is a general mechanism for production of force in cell biology (1). Spatially controlled polymerization of actin is responsible for a large variety of changes in cell shape leading to cell movement, like the protrusion of the lamellipodium (2), or the intracellular propulsion of vesicles (3, 4) and pathogens (5, 6). Actin assembly in these processes is controlled by various proteins present in the cytoplasm, which have been identified over the last 10 years (see refs. 7 and 8 for review). It is now well established that the Arp2͞3 complex associates with Wiskott-Aldrich Syndrome protein (WASp) family proteins to create new filaments locally by branching them (9), thus generating the dendritic organization of the actin array (10, 11). Because pure actin treadmilling is too slow to account for polymerization speed observed in these systems, this process has to be speeded up by regulatory proteins (8). One experimental model that has been used widely for the identification of these proteins is the bacteria Listeria monocytogenes, which are propelled inside cells by actin polymerization. The minimal subset of proteins necessary and sufficient to reconstitute the Listeria movement has been identified (12). In addition to Arp2͞3 and actin, the regulatory proteins ADF͞ cofilin, profilin, and a capping protein are necessary to enhance the efficiency of treadmilling (13) and control the life time and average length of filaments (14, 15). The essential role of these proteins has been confirmed recently (16) in vivo by using a RNA interference strategy.Biomimetic systems are useful for analyzing the physical aspects of actin-based motility. Listeria movement can be mimicked by fun...

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mentioning

confidence: 73%

Forces generated during actin-based propulsion: A direct measurement by micromanipulation

Marcy

Prost

Carlier

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

235

208

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“…Without additional chemical or mechanical cue, these cells are polarized, adherent and spontaneously motile. An active lamellipod, which extends in the cell front, is considered to act as the motor of the migration machinery via actin polymerization 10 . The central body is mainly occupied by the nucleus and at the cell rear, the uropod is a characteristic organelle of T lymphocytes whose role in cell migration remains enigmatic 11 .…”

Section: Resultsmentioning

confidence: 99%

Lymphocytes can self-steer passively with wind vane uropods

et al. 2014

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A wide variety of cells migrate directionally in response to chemical or mechanical cues, however the mechanisms involved in cue detection and translation into directed movement are debatable. Here we investigate a model of lymphocyte migration on the inner surface of blood vessels. Cells orient their migration against fluid flow, suggesting the existence of an adaptive mechano-tranduction mechanism. We find that flow detection may not require molecular mechano-sensors of shear stress, and detection of flow direction can be achieved by the orientation in the flow of the non-adherent cell rear, the uropod. Uropods act as microscopic wind vanes that can transmit detection of flow direction into cell steering via the on-going machinery of polarity maintenance, without the need for novel internal guidance signalling triggered by flow. Contrary to chemotaxis, which implies active regulation of cue-dependent signalling, upstream flow mechanotaxis of lymphocytes may only rely on a passive self-steering mechanism.

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“…2D), a 15% curvature bias corresponds to a radius of curvature of 2.3 μm and a bending energy of 0.6 k B T per μm of ATPbound filament (40). This amount of curvature could result from a lateral force of 1 pN applied perpendicularly to the end of a 0.05-μm-long filament fixed at the other end (44), which reflects the average force per filament due to membrane tension and rigidity (45,46) and the approximate length of free F-actin (47) at the leading edge of the cell. If the length of free F-actin is longer at the leading edge (48,49), the filaments require even less force to bend.…”

Section: A Fluctuation Gating Model For Branching By the Arp2/3 Complmentioning

confidence: 99%

Actin filament curvature biases branching direction

Risca

Wang²,

Chaudhuri

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

170

149

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Mechanical cues affect many important biological processes in metazoan cells, such as migration, proliferation, and differentiation. Such cues are thought to be detected by specialized mechanosensing molecules linked to the cytoskeleton, an intracellular network of protein filaments that provide mechanical rigidity to the cell and drive cellular shape change. The most abundant such filament, actin, forms branched networks nucleated by the actin-related protein (Arp) 2/3 complex that support or induce membrane protrusions and display adaptive behavior in response to compressive forces. Here we show that filamentous actin serves in a mechanosensitive capacity itself, by biasing the location of actin branch nucleation in response to filament bending. Using an in vitro assay to measure branching from curved sections of immobilized actin filaments, we observed preferential branch formation by the Arp2/3 complex on the convex face of the curved filament. To explain this behavior, we propose a fluctuation gating model in which filament binding or branch nucleation by Arp2/3 occur only when a sufficiently large, transient, local curvature fluctuation causes a favorable conformational change in the filament, and we show with Monte Carlo simulations that this model can quantitatively account for our experimental data. We also show how the branching bias can reinforce actin networks in response to compressive forces. These results demonstrate how filament curvature can alter the interaction of cytoskeletal filaments with regulatory proteins, suggesting that direct mechanotransduction by actin may serve as a general mechanism for organizing the cytoskeleton in response to force.actin-based motility | autocatalytic branching | bending fluctuations | worm-like chain | force sensing

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The Actin-Based Nanomachine at the Leading Edge of Migrating Cells

Cited by 247 publications

References 57 publications

Forces generated during actin-based propulsion: A direct measurement by micromanipulation

Forces generated during actin-based propulsion: A direct measurement by micromanipulation

Lymphocytes can self-steer passively with wind vane uropods

Actin filament curvature biases branching direction

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